Antibodies and T-cell receptors (TCR) are the two key molecules of the adaptive immune system in vertebrates having the unique ability to bind and recognize specific structural elements—so called antigens. Antibodies and TCRs are members of the immunoglobulin superfamily of proteins, a family which is characterized by the presence of a immunoglobulin domain or fold in their 3-dimensional protein structure. Antibodies are usually produced by blood cells in either soluble or membrane bound form (B-cell receptor) and aid the immune system in the detection and targeting of potentially pathogenic structures within the host organism—usually structures of pathogenic organisms like bacteria or viruses. TCRs on the other hand are expressed on T-cells and mediate the recognition of antigens presented by the major histocompatibility complex (MHC), among many others antigens which are specific for tumor cells. Due to their key function in the host's detection of potential threads, they are central in the development of immune system based therapeutics for the treatment of many diseases including cancerous and infectious diseases.
The T cell receptor is expressed on the cell surface of T cells as part of the adaptive immune system and is capable of recognizing peptide antigens presented on MHC class I and II molecules. The binding of the cognate antigen is followed by a cytolytic or cytokine secretion response, respectively, that promotes the elimination of cognate antigen presenting cells. In immunotherapy, this strategy may be employed to eradicate aberrant cells that present tumor associated antigens to cure cancer diseases (Rosenberg et al., 2008). Under certain circumstances, tumor-reactive T cells will not be identified in tumor patients due to functional unresponsiveness or low T cell frequencies. In order to circumvent central and peripheral tolerance mechanisms one aims at providing tumor-reactive high-affinity (TCRs) by genetic reprogramming of the patient's T cells. For this, the TCR encoding sequences are cloned into an e.g. retroviral shuttle vector. The cells are isolated from peripheral blood and treated with the retro:virally recombinant particles harvested from the supernatant of a producer packaging cell line. The heterologous expression of this TCR can be monitored by flow cytometry analysis. Afterwards, the reprogrammed autologous T cells are expanded short-term ex vivo and eventually are adoptively transferred to the patient. This therapeutical concept proved to be effective in the regression of progressive cancer diseases in few clinical trials recently (Johnson et al., 2009).
However, the T cell receptor consists of an αβ-heterodimer each chain comprising the antigen-recognizing variable Vα- or Vβ- and a constant Cα- or Cβ-domain, respectively, of an immunoglobulin-like fold linked by a natural disulfide bond close to the cell membrane. Both chains recognize specifically the MHC/peptide-complex via their CDR1-3 loops, located on each chain and cumulatively contribute to the affinity of the TCR. The expression of a second TCR in a human T cell may allow for the mixed pairing of exogenous and endogenous TCR chains to produce mis-paired or hybrid TCRs with unforeseen, potentially auto-reactive antigen specificities (Schumacher, 2002). The so-called “off-target”-reactions of reprogrammed T cells could be demonstrated in a mouse model leading to overt auto-immune disease, such as symptoms of so-called graft-versus-host disease (Bendle et al., 2010).
A reasonable strategy is to generate 3-domain single chain TCRs, which harbour covalently linked variable domains, that in theory hardly dissociate and mis-pair (FIG. 1A). The membrane anchoring of Vα-Linker-Vβ is accomplished by a constant Cβ-domain right after the variable Vβ-domain. In the past, this construct turned out to be functional only when the missing Cα-domain is co-expressed (DE10259713.8) and when the C-domains were murinized (DE102006041455.1). For this, the immunoglobulin-like folded Cα-domain has been modified to a membrane secretion protein by preceding it with a eukaryotic signal peptide (SP) (Voss et al., 2010).
The required murinization of a scTCR- or scCAR-construct coexpressed with a murine Cα-domain strengthen the interaction between the C-domains and thus, the stability of the TCR or CAR-construct. The classical scTCR or scCAR-scaffold, that is based on the fusion of the CD3ζ-signaling domain at the expense of Cα does not require murinization. The novel disulfide bonds may be applied to both strategies in order to improve the stability of the variable scTCR- or scFv-fragment. Due to structural homology of αβTCRs and γδTCRs, the same disulfide bonds may also be applied to stabilize scTCR-fragments of a γδTCR. In the meantime minimal murinization strategies allow for the substantial reduction of xenogeneic moieties to avoid immunogenic reactions against the chimeric molecule (Bialer et al., 2010; Sommermeyer and Uckert, 2010). For this, 9 murine amino acid residues in Cα and Cβ are sufficient at all to trigger the beneficial effect of stabilizing scTCR or scCAR-expression. Importantly these residues are located at the inner interface of the C-domains and thus, will be hardly accessible to the host immune system, in particular the complement system, to initiate any host versus graft immune response.
In a single or double chain format, antigen-recognizing fragments may also be derived from other species such as rabbit, goat, rat. The V-domains can be almost completely humanized except the sequences CDR1-3 of either chain that are responsible for specific antigen recognition (for example in the known antibody Herceptin®).
The introduction of an artificial disulfide bond at the interface of the C-domains at Cα T84C/Cβ S79C (Kuball et al., 2007) according to the nomenclature of the IMGT database (Lefranc, 2003; Lefranc et al., 2005) improved expression of the scTCR/Cα scaffold (Voss et al., 2010). Most importantly, this disulfide bond does not affect the interaction strength of the Vα/Vβ-domains.
TCR gene therapy is at the beginning of its clinical application and needs further basic research to proceed from “bench to bedside”. In experimental and animal models, an alternative strategy to avoid mis-pairing utilizes the i) 4-domain scTCR concept, in that the signalling component CD3ζ including its transmembrane region of the CD3-complex is fused to the aforementioned 3-domain scTCR (Willemsen et al., 2000). However, the coupling to the signalling component CD3ζ may induce its over-expression with unforeseen consequences on antigen specificity and T cell signalling.
Another strategy is to include an artificial disulfide bond, that covalently links the Cα- with the Cβ-domain (WO2004/033685). This approach resulted originally from experiments to increase the stability of bacterially expressed soluble double chain (dc) TCRs for protein crystallization (Boulter et al., 2003) and has been generalized to phage display for the isolation of high-affinity tumor-reactive TCR in vitro (Avidex, Abingdon, UK; (Li et al., ZOOS)). Disulfide bridges form lately during the posttranslational modification steps after ribosomal protein biosynthesis in the endoplasmic reticulum. Although dcTCRs pair earlier in the endoplasmic reticulum, the introduction of an artificial disulfide bond in C-domains shifts the equilibrium of chain pairing towards dcTCRs being able to create the cysteine bridge. However, this does not inhibit pairing of exogenous and endogenous chains, due to the stereochemical similarity of serine and threonine. Additionally, cysteine is a rather small polar amino acid that may allow approximation of almost all amino acid residues of the interacting TCR chain.
The introduction of reciprocal mutations, that sustain the steric and electrostatic environment at the interface of constant domains, modified complementarity such that almost exclusively the exogenous TCRs fit together, thereby avoiding the formation of hybrid TCR (Voss et al., 2008). In C-domains reciprocally mutated TCRs are almost as effective as wild type TCRs in effector function in vitro (Voss et al., 2008) and in vivo albeit showing a reduced structural avidity in tetramer analysis. This makes it difficult to monitor and to track them in vivo. Additionally, they do not entirely avoid formation of hybrid TCRs. The potential to avoid mis-pairing probably relates to the intrinsic qualities of TCR subfamilies and CDR3 sequences that contribute to TCR chain pairing (Heemskerk et al., 2007)
Further on, specific pairing of the exogenous TCRs is believed to be improved by the murinization of the constant domains of double chain TCRs (Voss et al., 2006; Cohen et al., 2006). It is shown, that murine constant domains have a higher affinity to each other due to more pronounced basic patches at the interface of Cβ that binds more strongly to Cα by electrostatic interactions. Recently, it was shown that murinization could be confined to a minimal set of defined residues so as to minimize xenogeneic reactions (Bialer et al., 2010; Sommermeyer and Uckert, 2010). The preferential pairing and thus, themodynamic and proteolytic stabilization of murinized TCRs outcompetes the surface expression of endogenous TCRs since endogenous and exogenous TCRs have to compete for binding to the CD3-complex as a prerequisite for secretion to the cell surface. Additionally, murine constant domains seem to efficiently interact with human CD3 components, as this in general is an elementary step in T cell signaling (Call et al., 2002). However, murine C-domains are still able to pair with endogenous C-domains despite their differences and hence may still lead to unwanted mis-pairing.
The codon-optimization of human and murine TCR substantially increased expression and functionality of heterologously expressed TCR in human T cells. The strategy yielded improved translation due to usage of the most frequent triplets in eukaryots, the adaptation of the GC-content, the elimination of cryptic splice-donor sites in RNA sequences and the avoidance of repetitive sequences, killer motifs and RNA secondary structure ((Jorritsma et al., 20C17); GENEART, Regensburg). However, it is by far not as efficient as murinization of TCRs to outcompete and down-regulate endogenous TCR expression. Additionally, it does not evoke preferential pairing of exogenous TCRs on a molecular basis. Therefore, mis-pairing of TCR chains still occurs.
For a short time, there are efforts to specifically eliminate endogenous TCRs by the digestion of the TCR RNA pool through siRNAs (Okamoto et al., 2009) or to even excise the related genes from the genome by zinc finger nucleases (Sangamo, Richmond, Calif.; (Miller et al., 2007)) Some of these strategies have a minimal impact on TCR structure and immunogenicity and additionally did not yield full knock-out of endogenous TCRs. At present genome editing is at its experimental advent and quite far away from clinical application.
Recent experiments demonstrated that a human WT1 (Xue et al., 2010)—and a CMV-specific scTCR were non-functional although they were designed to be co-expressed with murine Cα the same way as another, fully functional human scTCR gp100 (Voss et al., 2010): They were murinized in C-domains, modified to form the artificial disulfide bond (Kuball et al., 2007) and even codon-optimized (Jorritsrna et al., 2007) to increase expression. Secondly, recent hints indicated that residual mis-pairing with endogenous TCR chains still took place although it was argued in literature (Chung et al., 1994) that this should have been completely avoided by sterical hindrance of the Vα-moiety in scTCR-design.